Once thought to be primarily a carrier molecule of genetic information, RNA is now known to perform a wide variety of important biological tasks, including enzymatic catalysis, molecular recognition and gene regulation. This diverse functionality often depends on the folding of RNA into compact, three-dimensional (3D) structures and obtaining accurate knowledge of RNA 3D structure can be essential for understanding RNA function. Determining RNA structure via X-ray crystallography or NMR, however, is far more difficult than RNA sequencing and only a small fraction of RNA sequences currently have known structural models. Objective and Relevance: Computation offers a unique avenue to explore whether RNA folds not yet experimentally observed exist and, if so, whether new RNA functionality can be uncovered. In order to address these questions, this proposal aims to computationally engineer a novel RNA sequence capable of adopting a de novo three- dimensional, tertiary fold. If successful, this work would represent a rigorous proof-of- principle of our current understanding of the relationship between RNA sequence and structure. Further, it would develop computational tools that could be used to design and classify RNAs with novel folds or functions, opening the door to the development of potentially novel RNA therapeutics or molecular scaffolds. Specific Aims: (I) To computationally generate novel, 3D RNA backbone structures; (II) to computationally predict nucleotide sequences able to adopt the engineered RNA backbone folds; and (III) to experimentally characterize the tertiary structure of de novo designed RNA sequences and search genomes for natural RNA counterparts Study Design: A computational tool to create novel 3D RNA folds will be developed by use of a simplified RNA backbone representation and a knowledge-based energy function. Sequences will be designed for promising novel globular RNA folds by a variety of available tools, including isostericity matrices specifically developed with the RNA backbone geometry in mind. Hydroxyl radical foot-printing will then be used to probe whether designed sequences adopt de novo tertiary structures consistent with the engineered fold and X-ray crystallography of the most promising fold will be attempted. Finally, the designed, RNA fold will be used to search genomes for any naturally occurring RNA counterparts to the de novo RNA fold.